CDKN2a Companion Diagnostic for Bladder Cancer Interferon Therapy

ABSTRACT

A method of treating a human with bladder cancer that is not responsive to treatment with bacillus Calmette-Guérin, comprising measuring the human&#39;s level of CDKN2A expression, and then instilling into the human an agent which induces interferon expression, e.g., interferon polypeptide or a gene therapy vector carrying an interferon transgene.

RELATED APPLICATIONS

This application is a United States National Stage filing ofPCT/US2018/064829 filed 11 Dec. 2018, which in turn asserts priorityfrom United States provisional patent filing Ser. No. 62/597473 filed 12Dec. 2017, the contents of which are here incorporated by reference.

UNITED STATES GOVERNMENT FUNDING/INTEREST

None

BACKGROUND

Non-muscle-invasive bladder cancer (NMIBC) represents the most commondisease state for patients with newly diagnosed bladder cancer. Thosewith high-grade (HG) tumors are at significant risk for both recurrenceand progression. Bacillus Calmette-Guerin (BCG) represents the currentpreferred management. Nonetheless, approximately 30% of patients willnot respond to BCG; among those who demonstrate an initial response,more than 50% will experience recurrence and progression duringlong-term follow-up.

The optimal management of patients with persistent or recurrent tumorafter BCG remains controversial. Although radical cystectomy providescancer eradication, many patients are elderly, have significantco-morbidities with an attendant diminished performance status, andoften are unwilling to undergo radical extirpative surgery.Non-extirpative treatment options are available, but studies to datehave included relatively small patient numbers and used varieddefinitions of treatment success. Indeed, the US Food and DrugAdministration (FDA) and genitourinary oncology community agree thatscant progress has been made in the management of this disease since theinitial approval of BCG. Thus an effective alternative to radicalcystectomy for patients with disease recurrence after BCG treatmentremains an important unmet clinical need.

Several agents have been evaluated as second-line treatment after BCG;however, none (to date) have provided robust and durable responses.Valrubicin (Valstar; Endo Pharmaceuticals, Malvern, Pa.), the only agentcurrently approved by the FDA for the treatment of BCG-refractory CIS,provided a complete response rate of 18% at 6 months and a 1-yeardisease-free survival rate of approximately 10%. Promising results fromearly-phase trials have been reported for intravesical taxane andgemcitabine. Joudi et al. reported the final results from a nationalmulticenter phase II trial of BCG plus IFNα-2b and noted that 45% ofpatients with BCG failure were free from recurrence at 2 years. However,only 44% were treated for an HG recurrence, and 61% received only oneprior course of BCG. A recent retrospective analysis of BCG and IFNα-2breported a 38.6% RFS at 12 months. Again, many of these patients (20 of44) received only one prior course of BCG, and 16 patients experiencedrelapse after 12 months. Overall, the limited number of patients studiedin previous trials, as well as the modest RFS with treatment despite aless stringently defined eligibility, illustrates the unmet need foreffective and evidence-based second-line therapy for patients withBCG-unresponsive disease that improves disease-specific patient outcomesand avoids cystectomy.

Recombinant intravesical interferon alfa-2b protein (IFNα-2b; Intron A;Merck, Kenilworth, N.J.) demonstrated promising initial clinical resultsin NMIBC.

Intravesical IFNα-2b gene delivery offers a novel approach and increasesthe duration of exposure to IFNα-2b. Recombinant adenovirus(rAd)-IFNα-2b is a replication-deficient adenovirus-based gene transfervector that encodes the human IFNα-2b gene. Syn3, a polyamidesurfactant, is incorporated into the drug formulation (ADSTILADRIN®rAd-IFNα/Syn3™, FKD Therapies Oy, Kuopio, Finland) to enhance adenoviraltransduction of the bladder lining. Dramatic enrichment of rAd-IFNα genetransfer and expression has been shown with Syn3™ in both normalurothelium and human urothelial carcinoma that grows in mice.rAd-IFNα-2b gene therapy mimics the physiologic events associated withviral infection, which results in local rather than systemic IFNα-2bproduction and subsequent tumor regression.

We performed a phase I dose-ascending study of rAd-IFNα/Syn3 forpatients with BCG-refractory and relapsing NMIBC. First-generationreplication-deficient serotype 5 adenovirus vector, which expressedhuman interferon alfa-2b (IFNα-2b) cDNA under a cytomegaloviruspromoter, was produced under good manufacturing practice conditions in293 cells, as previously described, with slight modifications of theprocess. It was tested to be free of endotoxin, microbiologiccontaminants, and other impurities. The structure of the vector wasverified by sequencing. Production of recombinant IFNα-2b was verifiedfrom each production lot with immunologic methods. The excipient Syn3 isa polyamide surfactant that enhances adenoviral gene transfer to thebladder epithelium. Dose-dependent adenoviral gene transfer and urineconcentrations of IFNα-2b were confirmed. Of 14 patients treated withdose levels of rAd-IFNα/Syn3® that resulted in measurable urine IFNα,six (43%) were free from recurrence at 3 months and had no dose-limitingtoxicity, and two patients remained disease free at 29 and 39 months.

These provocative findings, predominantly at the two highest doses,prompted us to pursue a phase II study, designed to evaluate theefficacy and safety of intravesical rAd-IFNα/Syn3™ for patients with HGNMIBC refractory to, or with relapse after, BCG. This randomized,open-label, parallel-arm study was conducted across 13 centers in theUnited States between Nov. 5, 2012, and Apr. 8, 2015. The protocol,administrative oversight, and accrual timelines were designed andconducted by the Society of Urologic Oncology Clinical TrialsConsortium. The study protocol and informed consent form were reviewedand approved by the respective responsible site institutional reviewboards and biosafety committees.

We assessed the efficacy and safety of recombinant adenovirus interferonalfa with Syn3™ (rAd-IFNα/Syn3™), a replication-deficient recombinantadenovirus gene transfer vector, for patients with high-grade (HG)BCG-refractory or relapsed NMIBC. In this open-label, multicenter (13centers), parallel-arm, phase II study, of 43 patients with HGBCG-refractory or relapsed NMIBC received intravesical rAd-IFNα/Syn3™(randomly assigned 1:1 to 1×1011 viral particles (vp)/mL or 3×1011vp/mL). Patients who responded at months 3, 6, and 9 were retreated atmonths 4, 7, and 10. The primary end point was 12-month HGrecurrence-free survival (RFS). All patients who received at least onedose were included in efficacy and safety analyses.

In our study, forty patients received rAd-IFNα/Syn3™ (1×1011 vp/mL,n=21; 3×1011 vp/mL, n=19) between Nov. 5, 2012, and Apr. 8, 2015. Thetrial was designed to enroll 40 patients unable or unwilling to undergoradical cystectomy, and there were two dosage groups of 20 patientseach. Eligible patients were 18 years or older and had HG BCG-refractoryor relapsed NMIBC, including papillary NMIBC alone (Ta or T1), carcinomain situ (CIS) alone, or a combination of CIS and papillary disease.BCG-refractory disease was defined as the inability to achieve adisease-free state at 6 months after adequate induction BCG therapy witheither maintenance or reinduction at 3 months. “Adequate induction” wasdefined as a minimum of five of six treatments, and “adequatemaintenance” was defined as a minimum of two of three treatments. BCGrelapse was defined as recurrence within 1 year after a completeresponse to adequate BCG treatment (at least five and twoinstillations). Patients were required to have undergone visuallycomplete resection of papillary lesions by transurethral resection ofbladder tumors. Patients could not have received intravesical therapywithin 3 months before beginning study treatment, with the exception ofcytotoxic agents when administered as a single instillation immediatelyafter a transurethral resection. All participants who entered the studyprovided written or oral informed consent.

Patients were assigned by computer-generated random assignment, with aconstrained 1:1 sequence, to receive either low-dose (1×1011 viralparticles [vp]/mL) or high-dose (3×1011 vp/mL) rAd-IFNα/Syn3. Thesedoses were the most promising observed in the phase I study. The totaldoses administered were 7.5×1012 vp in the low-dose group and 2.25×1013vp in the high-dose group. Treatment allocation was performed centrallywith a block size of two for all patients who had successfully completedscreening, with the constraint that the first four patients at each sitewere balanced between cohorts.

rAd-IFNα/Syn3™ in 75 mL was administered intravesically through aurethral catheter, with a planned retention time of 1 hour; ananti-cholinergic treatment was allowed to relieve urinary urgency andpermit adequate retention. Patients without recurrence of HG disease atmonths 3, 6, and 9, as evaluated by cytology, cystoscopy, and biopsy (ifclinically indicated) were then retreated at months 4, 7, and 10. At 12months, a final efficacy evaluation was performed. This evaluationincluded a protocol-mandated biopsy from the site of the index tumor andat least five random biopsies, including the bladder dome, trigone,right and left lateral wall, posterior wall, and prostatic urethra inmen with positive cytology or prior disease in this region.

During the study, patients were contacted weekly by phone for the firstmonth after each treatment on days 7, 14 (of months 7 and 10 only), 21,and 28 (±1 day) to provide information about adverse events (AEs) andconcomitant medication use. Assessments for treatment failure were madebetween 14 and 7 days before retreatment. Patients who were withdrawnfrom treatment before study completion underwent a safety assessment atleast 30 days after last administration of the study drug. All patientsare being monitored in a 3-year long-term follow-up period to (1)determine recurrence of HG disease in those patients with a completeresponse and (2) to assess the long-term impact of treatment withrAd-IFNα/Syn3™

The primary end point was freedom from HG disease recurrence at 12months, defined by a negative for cause or end of study biopsy.Secondary end points included response to treatment, defined as noevidence of recurrence of HG disease at 3, 6, and 9 months; incidenceand time to cystectomy; and concentration of IFNα-2b in the urine.Safety assessments included physical examination, monitoring of vitalsigns, ECG, and standard clinical chemistry, hematology, and urinalysisassessments (performed by local laboratories). Safety end points includetype, incidence, relatedness, and severity of AEs and severe (≥grade 3)AEs (SAES), as assessed by National Cancer Institute Common TerminologyCriteria for Adverse Events (version 4.03).

We determined that a cohort of 20 patients would be sufficient to givean 80% probability of rejection of a HG recurrence-free survival (RFS)rate of 10% with an exact 5% one-sided test when the true HG RFS ratewas 35%. The operating characteristics for this Fleming design werecalculated exactly with the binomial distribution described by A′Hern.The hypothesis—that the response rate was equal to or less than thereference rate-was rejected if five or more of the 20 patients achievedHG RFS at 12 months. The proportion of patients who achieved HG RFS at3, 6, 9, and 12 months was reported for each dose group, together withan exact 90% CI for the proportion. The time to HG recurrence or deathwas summarized with the Kaplan-Meier method. Analyses were performedwith SASTM (version 9 or later; SAS Institute, Cary, N.C.). Both thesafety and efficacy (modified intention-to-treat) analysis sets includedall patients who received at least one dose of rAd-IFNα/Syn3®. A datamonitoring committee oversaw the study according to the data monitoringplan. All analytical assays were developed and validated. Samples weretested according to good laboratory practices methods at CovanceLaboratories Ltd (Harrogate, United Kingdom).

Baseline patient characteristics are provided in Table 1:

TABLE 1 Baseline Patient Characteristics No. (%) by rAd-IFNα/Syn3 DoseGroup 1 × 10¹¹ 3 × 10¹¹ No. (%) vp/mL vp/mL Overall Characteristics (n =21) (n = 19) (N = 40) Median (IQR) age, years 70 (67-74) 73 (62-81) 70.5(64.5-77.5) Sex Male 19 (90) 14 (73.7) 33 (82.5) Female 2 (9.5) 5 (26.3)7 (17.5) ECOG PS   0 16 (76.2) 18 (94.7) 34 (85.0)   1 5 (23.8) 1 (5.3)6 (15.0) History of radiation therapy* 1 (4.8) 1 (5.3) 2 (5) BCG failureclassification Relapsed 10 (47.6) 9 (47.4) 19 (47.5) Refractory 11(52.4) 10 (52.6) 21 (52.5) No. of previous BCG courses   1 1 1 2   2 1012 22 ≥3↑ 10 6 16 Primary tumor classification at enrollment CIS 12(57.1) 9 (47.4) 21 (52.5) Ta 2 (9.5) 2 (10.5) 4 (10) Ta and CIS 3 (14.3)1 (5.3) 4 (10) T1 2 (9.5) 4 (21.1) 6 (15) T1 and CIS 2 (9.5) 3 (15.8) 5(12.5) Abbreviations: BCG, bacillus Calmette-Guerin; CIS, carcinoma insitu; ECOP PS, Eastern Cooperative Oncology Group performance status;IQR, interquartile range; rAd-IFNα/Syn3, recombinant adenovirusinterferon alpha protein/Syn3 (a nonreplicating recombinant adenovirusgene transfer vector for patients with high-grade BCG-refractory orrelapsed non-muscle-invasive bladder cancer); Ta, papillary urothelialcarcinoma confined to the mucosa; T1, micro-invasive urothelialcarcinoma invasive into lamina propria but not muscularis propria; vp,viral particles. *Radiology was 10 or more years before screening ineach of these three patients; as such, they were deemed eligible forstudy enrollment. ↑Range of previous courses, 3 to 8.

The 12-month HG RFS rate was comparable between the two dose groups,with 33.3% of patients (7 of 21; 90% CI, 16.8 to 53.6) in the low-dosegroup and 36.8% (7 of 19; CI, 18.8 to 58.2) in the high-dose group aliveand free of HG disease at 12 months. Overall, 35.0% of patients (14 of40; 90% CI, 22.6% to 49.2%) remained free of HG recurrence at 12 monthsafter the initiation of rAd-IFNα/Syn3 treatment. Off-schedule diseaseassessments did not affect findings (Appendix, online only). The mediantime to HG recurrence or death was 6.5 months (90% CI, 3.52 to 12.78months); the median time to HG recurrence was 3.52 months (90% CI, 3.02to 12.78 months) for the low-dose group and was 11.73 months (90% CI,5.88 months to not evaluable) for the high-dose group.

Fourteen patients (35.0%; 90% CI, 22.6% to 49.2%) remained free of HGrecurrence 12 months after initial treatment. Comparable 12-month HG RFSwas noted for both doses. Of these 14 patients, two experiencedrecurrence at 21 and 28 months, respectively, after treatmentinitiation, and one died as a result of an upper tract tumor at 17months without a recurrence. rAd-IFNα/Syn3 was well tolerated; no gradefour or five adverse events (AEs) occurred, and no patient discontinuedtreatment because of an adverse event. The most frequently reporteddrug-related AEs were micturition urgency (n=16; 40%), dysuria (n=16;40%), fatigue (n=13; 32.5%), pollakiuria (n=11; 28%), and hematuria andnocturia (n=10 each; 25%).

When patient subgroups and secondary end points were considered inexploratory analyses, the 12-month HG RFS rates were broadly similar formen and women, for younger and older patients, for refractory orrelapsed NMIBC, for CIS only or papillary tumors and CIS, and forpatients with Ta and T1 disease only.

TABLE 2 Incidence of HG RFS at 3, 6, 9 and 12 Months rAd-IFNα/Syn3 DoseGroup 1 × 10¹¹ vp/mL 3 × 10¹¹ vp/mL Overall (n = 21) (n = 19) (N = 40)No. (%) No. (%) No. (%) of 90% Cl of 90% Cl of 90% Cl Variable Patients(%)* Patients (%)* Patients (%)* RFS at secondary end point analysistime 3 months 10 (47.6)  28.6-67.2 13 (68.4)  47.0-85.3 23 (57.5) 43.3-70.8 6 months 8 (38.1) 20.6-58.3 9 (47.4) 27.4-68.0 17 (42.5) 29.2-56.7 9 months 8 (38.1) 20.6-58.3 9 (47.4) 27.4-68.0 17 (42.5) 29.2-56.7 12 months 7 (33.3) 16.8-53.8 7 (36.8) 18.8-58.2 14 (35.0) 22.6-49.2 HG recurrence-free subgroup at 12 months Refractory NMIBC 8(38.1) 20.6-58.3 (n = 31) Relapsed NMIBC 6 (31.6) 14.7-53.0 (n = 19) CISonly 6 (28.6) 13.2-48.7 (n = 21) Papillary tumor 3 (33.3)  9.7-65.6 (n =9) Ta + Ti disease only 5 (50.0) 22.2-77.8 (n = 10) Serum antiadenoviralantibody Positive (n = 22) 10 (45.5)  27.1-64.7 Negative (n = 17) 4(23.5)  8.5-46.1 Abbreviations: CIS, carcinoma in situ; GG, high-grade;NMIBC, non-muscle-invasive bladder cancer; rAd-IFNα/Syn3, recombinantadenovirus interferon alpha protein/Syn3 (a nonreplicating recombinantadenovirus gene transfer vector for patients with high-grade bacillusCalmette-Guerin-refractory or relapsed NMIBC); RFS, relapse-freesurvival; Ta, papillary urothelial carcinoma confined to the mucosa; Ti,micro invasive urothelial carcinoma invasive into lamina propria but notmuscularis propria; vp, viral particles. *Cl is for the proportion ofpatients with HG RFS; 90% Cls are based on the exact binomial method.

Interestingly, of the 14 patients who were recurrence free at 12 months,10 (71%) of the 14 had an anti-adenovirus antibody response (defined asfour times the pre-dose titer), compared with 11 (24%) of 25 whoexperienced recurrence.

In long-term follow-up, seven patients (18%) who withdrew from the studybecause of HG disease recurrence within the 12-month study period diedat a median of 16 months (range, 2 to 26 months) after the withdrawaldate. There is no indication that these deaths were treatment related.The cause of death was unknown in four patients, whereas two died as aresult of progressive bladder cancer and one died as a result of liverfailure unrelated to treatment 17 months after withdrawal from thestudy. The four patients for whom the cause of death is unknown werebeing observed locally after they completed their end-of-studyevaluation. Fourteen patients (35%) who experienced an HG recurrencewithin the first year underwent a radical cystectomy at a median of 9months (range, 4 to 28 months) from day 1 of month 1.

Patients are being monitored for 3 years to collect long-term follow-updata. Of the 14 patients who remained disease free at 12 months,additional follow-up data are being collected for 11; 3 withdrew fromthe study. Nine of these 11 patients are alive, and eight remaineddisease-free during a period of 15 to more than 36 months. Two patientsexperienced HG recurrence at 21 and 28 months, respectively, from thestart of treatment. One of these patients who experienced progression tomuscle invasion underwent a radical cystectomy 31 months after theinitiation of treatment and later died at 41 months. The other, whoexperienced recurrence at 21 months, remained alive and free fromdistant recurrence at 36 months. One patient free from bladderrecurrence at 12 months died as a result of an upper tract tumor at 17months.

TABLE 3 Durability of HG RFS Since Start of Treatment WithrAd-IFNα/Syn3 ® Duration of Bladder Time of Last Stage at Dose HG RFSSince Follow-Up from Status at Last Entry Group Day 1 (months) Day1(months) Follow-Up Ta/CIS High 21 47 Recurrence of HGD Died at 38 monthsTa Low 28 41 Recurrence of 28 months Cystectomy at 31 months Died at 41months CIS Low 15 15 CR Withdrew Ta/CIS Low 30 36 Recurrence of HGD TaHigh 16 16 CR Withdrew T1/CIS Low 35 37 CR T1 Low 30 50 CR T1 High 36 36CR CIS High 38 39 CR CIS High 34 37 CR CIS High 27 27 CR CIS Low 34 37CR T1/CIS Low 17 17 Died of upper tract recurrence Ta High 13 13 CRWithdrew NOTE. Duration of HG RFS represent the number of months fromday 1 that a complete response within the bladder has been documentedbased on yearly report. Three patients withdrew from the study after the1-month end-of-study evaluation. Two patients had recurrence of HGD at21 and 28 months from day 1. One of these patients underwent acystectomy but later died. One patient died of an upper tract tumor witha bladder recurrence. Abbreviations: CIS, carcinoma in situ; CR,complete response; HG, high-grade; HGD, high-grade disease;rAd-IFNα/Syn3, recombinant adenovirus interferon alpha protein/Syn3 ® (anonreplicating recombinant adenovirus gene transfer vector for patientswith HG bacillus Calmette-Guerin-refractory or relapsednon-muscle-invasive-bladder cancer); RFS, relapse-free survival; Ta,papillary urothelial carcinoma confined to the mucosa; T1,micro-invasive urothelial carcinoma invasive into lamina propria but notmuscularis propria.

Our results showed that rAd-IFNα/Syn3™ was well tolerated. Itdemonstrated promising efficacy for patients with HG NMIBC after BCGtherapy who were unable or unwilling to undergo radical cystectomy.

While potentially promising, however, these data show several failingsin our treatment plan. First, treatment was ineffective in the majorityof patients: fully 65% of patients failed to achieve a 12-month HG RFSby intention-to-treat analysis of all patients dosed. Likewise, the12-month RFS in heavily pretreated patients was 31%. Notably, responseswere durable: the majority remained disease-free for close to 24 months.We noted that for patients with any element of CIS, 70% failed toachieve a durable complete response. Indeed, for patients with papillarydisease only at study entry, only 50% achieved RFS.

To address these failings, we have pursued two approaches. First, wehave designed and are currently pursuing a larger trial involvingsignificantly more patients and a high-dose of rAd-IFNα/Syn3™. Thistrial may provide evidence that a higher dose of rAd-IFNα/Syn3™ iseffective where a lower dose was not merely less effective, but entirelyineffective.

Second, we collected tissue samples from patients involved in theabove-discussed Phase II completed trial. These samples enable us toanalyze gene expression for each patient. This enables us to comparegene expression in patients who responded to treatment against geneexpression in patients who did not respond.

Gene expression analysis may be done using tumor biopsy samples.

Alternatively, for bladder cancer, urine samples if properly preservedmay contain exosomes which enable analysis of oncogene expression.Exosomes are small (30-100 nm) endocytic, cell-derived vesicles. Theyare secreted by most human cell types, including cancer cells, and theymay be absorbed via endocytosis into recipient cells. Exosomes cancontain functional bio-molecules such as dsDNA, and may be found inhuman blood and urine. Exosomal DNA (“exoDNA”) represents the entiregenome and, where the exosome is produced from a tumor cell, reflectsthe mutational status of the tumor cell. ExoDNA in tumor-derivedexosomes found in a urine sample taken from a bladder cancer patientthus provides a non-invasive circulating biomarker useful for thesensitive detection of cancer and a more accurate determination ofpotential responsiveness to interferon-based therapies.

ExoDNA includes both single-stranded and double-stranded DNA. ExoDNA isfound encapsulated in the interior of the exosome membrane and bound tothe exterior of the exosome membrane. In internal exoDNA, dsDNApredominates. Typically, exoDNA encapsulated in the interior of theexosome membrane ranges from 0.1-2.5 kb, while exoDNA bound to theexosome membrane exterior is >2.5 kb. Much of the exoDNA associated withtumor exosomes is double-stranded DNA bound to the exterior of theexosome membrane. ExoDNA, which includes dsDNA, can provide an unusuallyaccurate diagnostic tool because it provides a complete sample of thecomplete genome in readily-assayable form in the urine of a patient withbladder cancer.

Alternatively, one can assay micro vesicles. Micro vesicles are anothertype of extracellular vesicle, between 50 and 1,000 nanometers (nm) indiameter, found in many types of body fluids as well as the interstitialspace between cells. Micro vesicles are made from fragments of plasmamembrane. They are thus distinct from exosomes, which are smaller andgenerated intra-cellularly. In contrast to exosomes, which do notcontain mitochondrial DNA, micro vesicles derived from astrocytes andglioblastoma include mitochondrial DNA. Micro vesicles appear equivalentto exosomes for the purposes of this invention.

For a human patient diagnosed with bladder cancer, one may collectexosomes by collecting a urine sample. Sample storage techniques able topreserve DNA and RNA are known in the art. Similarly, isolation ofexosomes from urine may be done using conventional separationtechniques.

Alternatively, one may employ fluorescent in situ hybridization (FISH).FISH is a molecular cytogenetic technique that uses fluorescent probesthat bind to specific target parts of the chromosome, but whenadequately stringent hybridization conditions are used, does so with ahigh degree of sequence complementarity. FISH was developed in the early1980s. See e.g., Langer-Safer, P. R. et al., Immunological Method ForMapping Genes On Drosophila Polytene Chromosomes, 79 Proceedings of theNational Academy of Sciences 4381 (1982). FISH can detect and localizethe presence or absence of specific DNA sequences on chromosomes or inDNA fragments such as free DNA found in urine.

To use FISH in our diagnostic method, one constructs a probe able tohybridize with a portion of CDKN2A, and preferably with one or more ofthe eight exons contained in CDKN2A. The probe must be large enough tohybridize specifically with its target but not so large as to requirenon-stingent hybridization conditions, which conditions may lead toformation of false-positive hybridization to an inappropriate targetsequence. We prefer a probe of about 40 bp, but longer or shorter may beused depending on the specific CDKN2A mutation sought. Once made, theprobe is tagged directly with fluorophores, with targets for antibodiesor with biotin. Tagging can be done in various ways taught in the art,such as nick translation or Polymerase Chain Reaction using taggednucleotides.

To find out whether and where the fluorescent probe is bound to thetarget DNA in e.g., a urine sample, one may use fluorescence microscopy.FISH can also be used to detect and localize specific RNA targets (e.g.,mRNA) in cells, circulating tumor cells, and tissue samples.

As an alternative to sequencing exoDNA in a urine sample, one mayisolate free DNA from the urine sample and sequence that free DNA.

Analysis of exoDNA reveals a critical difference in the populations ofbladder cancer patients who respond to interferon therapy and those whodo not. Patients who respond to treatment have a different level ofexpression of CDKN2A than do patients who do not respond to treatment.These different levels in expression appear to arise from deletions inthe gene, or a loss of heterozygosity in the gene.

Our insight can improve the safety and efficacy of rAd-IFN gene therapybecause it enables the attending physician to limit treatment to thosepatients most likely to respond. Further, our insight can similarlyimprove the safety and efficacy of other instilled treatments whichinduce interferon expression, e.g., BCG vaccine. Similarly, our insightcan improve the effectiveness of cancer “checkpoint inhibitors.”Checkpoint inhibitors are thought to be ineffective in cancer patientswith CDKN2A deletions. Identifying such patients enables the artisan tocombine interferon therapy with checkpoint inhibitor therapy in suchpatients, using the interferon to overcome the inhibition of thecheckpoint inhibitor.

We thus here intend this patent to cover a method of treating bladdercancer, comprising diagnosing bladder cancer in a human, measuring thehuman's level of CDKN2A expression, and then instilling into the humanan agent which induces interferon expression.

We similarly intend this patent to cover that method of treating bladdercancer, where the bladder cancer is high-grade.

We similarly intend this patent to cover that method of treating bladdercancer, where the agent is a non-replicating agent such as rAd-IFN ormycobacterium cell wall extract.

We similarly intend this patent to cover that method of treating bladdercancer, where the agent is a replicating agent such as BCG vaccine.

We similarly intend this patent to cover that method of treating bladdercancer, where measuring the human's level of CDKN2A expression entailstaking a bladder tissue sample and measuring the level of CDKN2Aexpression in that tissue sample. Alternatively, we intend this patentto cover that method of treating bladder cancer, where measuring thehuman's level of CDKN2A expression entails taking a urine sample, andmeasuring the level of CDKN2A expression in it, preferably by analyzingthe exosomes in it, but also by analyzing e.g., free DNA in it.

We similarly intend this patent to cover that method of treating bladdercancer, where the human also gets treated with a cancer checkpointinhibitor.

We similarly intend our patent to cover a companion diagnostic test ableto measure the level of CDKN2A expression in a bladder tissue sample.

Further embodiments and variations will be readily apparent to theartisan after reviewing this disclosure. We thus intend our appendedlegal claims to encompass such embodiments.

We claim:
 1. A method of treating non-muscle invasive bladder cancer ina human, comprising: a. identifying a human diagnosed with non-muscleinvasive bladder cancer, and then b. measuring the human's level ofCDKN2A expression, and then c. instilling into the lumen of the bladderof said human interferon.
 2. The method of claim 1, where the bladdercancer is high-grade.
 3. The method of claim 1, where the interferon isadministered as a non-replicating vector.
 4. The method of claim 3,where said non-replicating vector comprises a replication-deficientviral vector carrying an interferon transgene.
 5. The method of claim 4,where said replication-deficient viral vector carrying an interferontransgene comprises nadofaragene firadenovec.
 8. The method of claim 1,where said measuring the human's level of CDKN2A expression entailstaking a bladder tissue sample and measuring the level of CDKN2Aexpression in that tissue sample.
 9. The method of claim 1, where saidmeasuring the human's level of CDKN2A expression entails taking a urinesample and measuring the level of CDKN2A expression in it.
 10. Themethod of claim 9, where said measuring the level of CDKN2A expressioncomprises analyzing the exosomes in said urine sample.
 11. The method ofclaim 9, wherein said measuring the level of CDKN2A expression comprisesanalyzing free DNA in said urine sample.
 12. The method of claim 1,further comprising the step of administering to said human a checkpointinhibitor after said step of measuring the level of CDKN2A expression.13. The method of claim 1, wherein said step of measuring the human'slevel of CDKN2A expression comprises using fluorescent in situhybridization using a probe able to hybridize with a portion of CDKN2A.14. The method of claim 13 wherein said probe comprises a portion whichhybridizes with at least one exon contained in CDKN2A.
 15. The method ofclaim 13 wherein said probe is about 40 bp in length.
 16. The method ofclaim 14 wherein said probe is about 40 bp in length.